Project Summary In graduate school I worked on cancer cell lines to identify interactions between sirtuin family demethylases and canonical and non-canonical Wnt signaling. To broaden my training, as a postdoctoral fellow I am studying intestine-specific gene expression, which reflects the outcome of extrinsic signals on cell-intrinsic mechanisms of transcriptional control. The intestine- restricted transcription factor CDX2 is required to maintain the activity of villus cell enhancers for intestine-specific gene expression. This application focuses on a novel class of ?anti-repressive? enhancers that depend critically on CDX2. I propose to establish the requirements of these enhancers in vivo (specific Aim 1), to define a molecular mechanism that distinguishes their particular activity (Aims 1 and 2), and to test the hypothesis that their activation is one limiting step in the differentiation of immature crypt progenitors into mature villus enterocytes (Aim 2). Trimethylation of Lysine 27 on Histone 3 (H3K27me3) is a histone mark that has been studied extensively for its repressive function at gene promoters but has not previously been implicated in regulation of distal enhancers in adult tissues. My preliminary data implicate a distinct subset of CDX2-dependent enhancers in regulation of several hundred intestinal genes and reveal that these ?anti-repressive? enhancers exclude H3K27me3 from extended genomic domains near some of the most CDX-dependent genes. In Specific Aim 1, I will use Eed-/- mice, which lack all H3K27me3, to test the specific hypothesis that excess H3K27me3 at ?anti-repressive? enhancers is directly responsible for curtailing gene expression. I will also use genome-wide mapping of the H3K27me3-specific demethylases KDM6A and KDM6B in wild-type and Cdx2-/- intestines to test the hypothesis that CDX2 functions at ?anti-repressive? enhancers to recruit these demethylases. In Specific Aim 2, I will extend these ideas to ask whether CDX2- dependent ?anti-repressive? enhancers are responsible for some of the largest increases in gene activity that occur when crypt progenitors mature into villus enterocytes. To this end, I will assess intestinal enhancers globally in crypt and villus cell fractions isolated from Atoh1-/- intestines, which house only enterocytes. Insights into the mechanisms that maintain intestinal homeostasis will improve understanding of disease, and the enhancer-associated epigenome- modifying enzymes described in this proposal are potential therapeutic targets in both cancer and regenerative biology. Moreover, by using state-of-the-art experimental and computational tools to test specific hypotheses, I will acquire expertise that will be invaluable in my transition from a mentored postdoctoral researcher to an independent investigator.